Protein products of frizzled genes are cell membrane receptors for Wnt proteins that play multiple roles during development. We examined the expression of nine frizzled genes in mouse chondrocytes, and detected transcripts of six of the nine genes. We also cloned the entire cDNA of mouse frizzled-1 and compared its cDNA sequence and the cysteine-rich and transmembrane domains of its translated product to sequences of frizzled-1 from C. elegans, Drosophila, chicken and human. We used the T31 Mouse/Hamster radiation hybrid panel to map the mouse frizzled-1 to mouse chromosome 5 (5 cM from the centromere), and frizzled-9 to mouse chromosome 5 (74 cM from the centromere).

The amnionless gene, essential for mouse gastrulation, encodes a visceral-endoderm-specific protein with an extracellular cysteine-rich domain.

Nat Genet. 2001; 27: 412-6

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Fate-mapping experiments in the mouse have revealed that the primitive streak can be divided into three functional regions: the proximal region gives rise to germ cells and the extra-embryonic mesoderm of the yolk sac; the distal region generates cardiac mesoderm and node-derived axial mesendoderm; and the middle streak region produces the paraxial, intermediate and lateral plate mesoderm of the trunk. To gain insight into the mechanisms that mediate the assembly of the primitive streak into these functional regions, we have cloned and functionally identified the gene disrupted in the amnionless (amn) mouse, which has a recessive, embryonic lethal mutation that interferes specifically with the formation and/or specification of the middle primitive streak region during gastrulation. Here we report that the gene Amn encodes a novel type I transmembrane protein that is expressed exclusively in the extra-embryonic visceral endoderm layer during gastrulation. The extracellular region of the Amn protein contains a cysteine-rich domain with similarity to bone morphogenetic protein (BMP)-binding cysteine-rich domains in chordin, its Drosophila melanogaster homolog (Short gastrulation) and procollagen IIA (ref. 3). Our findings indicate that Amn may direct the production of trunk mesoderm derived from the middle streak by acting in the underlying visceral endoderm to modulate a BMP signaling pathway.

Wnt-4 signaling plays a critical role in kidney development and is associated with the epithelial conversion of the metanephric mesenchyme. Furthermore, secreted Frizzled-related proteins (sFRPs) that can bind Wnts are normally expressed in the developing metanephros, and function in other systems as modulators of Wnt signaling. sfrp-1 is distributed throughout the medullary and cortical stroma in the metanephros, but is absent from condensed mesenchyme and primitive tubular epithelia of the developing nephron where wnt-4 is highly expressed. In contrast, sfrp-2 is expressed in primitive tubules. To determine their role in kidney development, recombinant sFRP-1, sFRP-2 or combinations of both were applied to cultures of 13-dpc rat metanephroi. Both tubule formation and bud branching were markedly inhibited by sFRP-1, but concurrent sFRP-2 treatment restored some tubular differentiation and bud branching. sFRP-2 itself showed no effect on cultures of metanephroi. In cultures of isolated, induced rat metanephric mesenchymes, sFRP-1 blocked events associated with epithelial conversion (tubulogenesis and expression of lim-1, sfrp-2 and E-cadherin); however, it had no demonstrable effect on early events (compaction of mesenchyme and expression of wt1). As shown herein, sFRP-1 binds Wnt-4 with considerable avidity and inhibits the DNA-binding activity of TCF, an effector of Wnt signaling, while sFRP-2 had no effect on TCF activation. These observations suggest that sFRP-1 and sFRP-2 compete locally to regulate Wnt signaling during renal organogenesis. The antagonistic effect of sFRP-1 may be important either in preventing inappropriate development within differentiated areas of the medulla or in maintaining a population of cortical blastemal cells to facilitate further renal expansion. On the other hand, sFRP-2 might promote tubule formation by permitting Wnt-4 signaling in the presence of sFRP-1.

Metallocarboxypeptidase Z (CPZ), a new member of the regulatory metallocarboxypeptidases, contains a 120-residue cysteine-rich region that has 20-35% amino acid sequence identity to Drosophila and mammalian frizzled proteins. In order to gain insights into the function of CPZ, we have examined the distribution of the protein by immunohistochemistry throughout mouse development. The expression of CPZ peaks at E9-E12, decreases in late gestation and falls further in adult tissues. CPZ expression in amnion cells, cochlear epithelial cells and surrounding mesenchyme, ventricular lining cells in the brain and cartilagenous condensations and surrounding connective tissue in ribs remains at high levels throughout mouse gestation. The expression pattern of CPZ overlaps with the expression pattern of several Wnt genes, consistent with the putative role of CPZ in Wnt signaling.

Drosophila epithelia acquire a planar cell polarity (PCP) orthogonal to their apical-basal axes. Frizzled (Fz) is the receptor for the PCP signal, and Dishevelled (Dsh) transduces the signal. Here, I demonstrate that unipolar relocalization of Dsh to the membrane is required to mediate PCP, but not Wingless (Wg) signaling. Dsh membrane localization reflects the activation of Fz/PCP signaling, revealing that the initially symmetric signal evolves to one that displays unipolar asymmetry, specifying the cells' ultimate polarity. This transition from symmetric to asymmetric Dsh localization requires Dsh function, and reflects an amplification process that generates a steep intracellular activity gradient necessary to determine PCP.

Structural, functional, and evolutionary characterization of novel members of the allatostatin receptor family from insects.

Biochem Biophys Res Commun. 2001; 282: 904-9

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By using degenerate primers based on known mammalian somatostatin receptors and the recently identified Drosophila allatostatin receptors (AlstR), we have cloned a novel receptor for the neuropeptide, allatostatin, from the cockroach Periplaneta americana. The receptor exhibits about 60% amino acid identity in the transmembrane regions when compared to the two known AlstRs from Drosophila melanogaster. In addition, two cDNA fragments were obtained from the stick insect Carausius morosus, one of which is similar to Drosophila AlstR, whereas the other is more similar to mammalian somatostatin receptors. Functional expression in Xenopus oocytes shows that the Periplaneta-AlstR exhibits high affinity to endogenous cockroach allatostatin peptides. Studies with synthetic peptides demonstrate that agonistic activity is mediated by the conserved C-terminal pentapeptide YXFGL-amide; in this sequence, amidation of the C-terminus is obligatory to maintain affinity. Thus, our studies provide a molecular basis for understanding the widespread biological activities of the allatostatin peptides. Copyright 2001 Academic Press.

Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains.

Nature. 2001; 412: 86-90

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Members of the Frizzled family of seven-pass transmembrane proteins serve as receptors for Wnt signalling proteins. Wnt proteins have important roles in the differentiation and patterning of diverse tissues during animal development, and inappropriate activation of Wnt signalling pathways is a key feature of many cancers. An extracellular cysteine-rich domain (CRD) at the amino terminus of Frizzled proteins binds Wnt proteins, as do homologous domains in soluble proteins-termed secreted Frizzled-related proteins-that function as antagonists of Wnt signalling. Recently, an LDL-receptor-related protein has been shown to function as a co-receptor for Wnt proteins and to bind to a Frizzled CRD in a Wnt-dependent manner. To investigate the molecular nature of the Wnt signalling complex, we determined the crystal structures of the CRDs from mouse Frizzled 8 and secreted Frizzled-related protein 3. Here we show a previously unknown protein fold, and the design and interpretation of CRD mutations that identify a Wnt-binding site. CRDs exhibit a conserved dimer interface that may be a feature of Wnt signalling. This work provides a framework for studies of homologous CRDs in proteins including muscle-specific kinase and Smoothened, a component of the Hedgehog signalling pathway.

Wnt glycoproteins have been implicated in diverse processes during embryonic patterning in metazoa. They signal through frizzled-type seven-transmembrane-domain receptors to stabilize beta-catenin. Wnt signalling is antagonized by the extracellular Wnt inhibitor dickkopf1 (dkk1), which is a member of a multigene family. dkk1 was initially identified as a head inducer in Xenopus embryos but the mechanism by which it blocks Wnt signalling is unknown. LDL-receptor-related protein 6 (LRP6) is required during Wnt/beta-catenin signalling in Drosophila, Xenopus and mouse, possibly acting as a co-receptor for Wnt. Here we show that LRP6 (ref. 7) is a specific, high-affinity receptor for Dkk1 and Dkk2. Dkk1 blocks LRP6-mediated Wnt/beta-catenin signalling by interacting with domains that are distinct from those required for Wnt/Frizzled interaction. dkk1 and LRP6 interact antagonistically during embryonic head induction in Xenopus where LRP6 promotes the posteriorizing role of Wnt/beta-catenin signalling. Thus, DKKs inhibit Wnt co-receptor function, exemplifying the modulation of LRP signalling by antagonists.

Seven-transmembrane receptors of the frizzled family can interact with secreted Wnt ligands and transmit Wnt signals into the cell. Dependent on the ligand receptor combination, distinct Wnt pathways are activated. Xenopus frizzled 7 (Xfz7) and Xwnt-8b as well as Human frizzled 5 (Hfz5) and Xwnt-5a can act synergistically in the activation of Wnt/beta-catenin target genes siamois (Xsia) and nodal related 3 (Xnr3) and in the induction of ectopic axes in Xenopus embryos. In order to characterize the role of different protein domains of Xfz7 in Wnt/beta-catenin signaling, chimeric Xfz7/Hfz5 receptors were generated in which the extracellular (N5-TC7) or the intracellular domains (NT7-C5) between Xfz7 and Hfz5 were exchanged. We present evidence that the extracellular domain of Xfz7 can interact with Xwnt-5a and that the intracellular C-terminus can transmit a Wnt/beta-catenin signal. Despite these abilities, Xfz7 and Xwnt-5a do not act synergistically in the activation of Wnt/beta-catenin targets. This implies that the interaction of a frizzled receptor with different ligands can result in distinct cellular responses.

Wnt signalling has an important role in cell fate determination, tissue patterning and tumorigenesis. Secreted antagonists of Wnt include Frizzled (Fz)-related proteins (FRPs), Cerberus, Wnt inhibitory factor (WIF) and Dickkopf (Dkk). FRPs, Cerberus and WIF have all been shown to act by binding and sequestering Wnt. We report a novel mechanism of Wnt-signalling inhibition by human Dkk-1. Dkk-1 demonstrated no interaction with Wnt but bound a single cell surface site with high affinity (K(D) = 0.39 nM). Its receptor was detectable in a complex with a relative molecular mass of 240,000 (M(r) 240K) with [(125)I] Dkk-1 by covalent affinity cross-linking. Wnt signalling through beta-catenin is mediated by the Fz receptor and a recently identified low-density-lipoprotein-receptor-related co-receptor, LRP6/Arrow. Overproduction of the 200K LRP6 protein, but not of Fz, strikingly increased Dkk-1 binding as well as the amount of the 240K cross-linked complex, which was shown to be composed of Dkk-1 and LRP6. Moreover, Dkk-1 function was completely independent of Fz but LRP6 dramatically interfered with the Dkk-1 inhibition of Wnt signalling. Thus, unlike Wnt antagonists, which exert their effects by molecular mimicry of Fz or Wnt sequestration through other mechanisms, Dkk-1 specifically inhibits canonical Wnt signalling by binding to the LRP6 component of the receptor complex.

Dickkopf proteins are secreted antagonists of the Wnt cell signalling molecules, which have a novel mode of action. Dickkopf1 binds to the LRP5/6 Wnt co-receptor and prevents the formation of active Wnt-Frizzled-LRP5/6 receptor complexes, thus blocking the canonical Wnt-beta-catenin pathway.

Identification and characterization of the epithelial polarity receptor "Frizzled" in Hydra vulgaris.

Dev Genes Evol. 2000; 210: 258-62

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The Wnt signaling pathway plays an important role in the specification of cell patterning during development in many species. Here we report the isolation and characterization of a putative Wnt receptor, Frizzled, in Hydra vulgaris. Analysis of the amino acid sequence of Frizzled in hydra reveals that this receptor contains many strong sequence similarities to other known Frizzled receptors. Hydra divergence is estimated to have occurred about one billion years ago; thus comparison of the Frizzled sequence of hydra with that of other species is likely to provide important information on the structure and function of those highly conserved regions. Northern and Southern blotting reveal that the Frizzled receptor in hydra has a 2.34-kb message size, and that it is encoded by a single gene. In situ hybridization using hydra frizzled as a probe reveals that the receptor message is restricted to the endoderm in adult hydra. This distribution supports the hypothesis that the Frizzled receptor is functioning in a pathway that controls cell differentiation in hydra.

Cloning and expression of the Wnt antagonists Sfrp-2 and Frzb during chick development.

Dev Biol. 2000; 218: 183-98

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The Wnt genes are known to play fundamental roles during patterning and development of a number of embryonic structures. Receptors for Wnts are members of the Frizzled family of proteins containing a cysteine-rich domain (CRD) that binds the Wnt protein. Recently several secreted frizzled-related proteins (Sfrps) that also contain a CRD have been identified and some of these can both bind and antagonise Wnt proteins. In this paper we report the expression patterns of the chick homologues of Frzb, a known Wnt antagonist, and Sfrp-2. Both genes are expressed in areas where Wnts are known to play a role in development, including the neural tube, myotome, cartilage, and sites of epithelial-mesenchymal interactions. Initially, Sfrp-2 and Frzb are expressed in overlapping areas in the neural plate and neural tube, whereas later, they have distinct patterns. In particular Sfrp-2 is associated with myogenesis while Frzb is associated with chondrogenesis, suggesting that they play different roles during development. Finally, we have used the early Xenopus embryo as an in vivo assay to show that Sfrp-2, like Frzb, is a Wnt antagonist. These results suggest that Sfrp-2 and Frzb may function in the developing embryo by modulating Wnt signalling.

The WD-repeat proteins are found in all eukaryotes and play an important role in the regulation of a wide variety of cellular functions such as signal transduction, transcription, and proliferation. Here we report on the cloning and characterization of a novel human WD-repeat gene, WDR6, which encodes a protein of 1121 amino acids and contains 11 WD-repeat units. WDR6 is unique since its 11 WD repeats are clustered into two distinct groups separated by a putative transmembrane domain. The WDR6 gene was mapped to chromosome 15q21 by fluorescence in situ hybridization. Northern analysis demonstrated that WDR6 is ubiquitously expressed in human adult and fetal tissues. WDR6 is not homologous to any previously identified human WD-repeat genes including WDR1 through WDR5. However, it was found to have significant sequence similarity with Arabidopsis thaliana hypothetical protein T7B11.12, yeast putative elongation factor G, and probable membrane protein YPL183c. All of them have been defined as WD-repeat proteins. Therefore, WDR6 is a novel protein and probably belongs to a highly conserved subfamily of WD-repeat proteins in which T7B11.12 and YPL183c are its distantly related members.

Molecular identification of a taste receptor gene for trehalose in Drosophila.

Science. 2000; 289: 116-9

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The molecular nature of sweet taste receptors has not been fully explored. Employing a differential screening strategy, we identified a taste receptor gene, Tre1, that controls the taste sensitivity to trehalose in Drosophila melanogaster. The Tre1 gene encodes a novel protein with similarity to G protein-coupled seven-transmembrane receptors. Disruption of the Tre1 gene lowered the taste sensitivity to trehalose, whereas sensitivities to other sugars were unaltered. Overexpression of the Tre1 gene restored the taste sensitivity to trehalose in the Tre1 deletion mutant. The Tre1 gene is expressed in taste sensory cells. These results provide direct evidence that Tre1 encodes a putative taste receptor for trehalose in Drosophila.

Molecular cloning and genomic organization of a second probable allatostatin receptor from Drosophila melanogaster.

Biochem Biophys Res Commun. 2000; 273: 571-7

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We (C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 269, 91-96) and others (N. Birgul et al. (1999) EMBO J. 18, 5892-5900) have recently cloned a Drosophila receptor that was structurally related to the mammalian galanin receptors, but turned out to be a receptor for a Drosophila peptide belonging to the insect allatostatin neuropeptide family. In the present paper, we screened the Berkeley "Drosophila Genome Project" database with "electronic probes" corresponding to the conserved regions of the four rat (delta, kappa, mu, nociceptin/orphanin FQ) opioid receptors. This yielded alignment with a Drosophila genomic database clone that contained a DNA sequence coding for a protein having, again, structural similarities with the rat galanin receptors. Using PCR with primers coding for the presumed exons of this second Drosophila receptor gene, 5'- and 3'-RACE, and Drosophila cDNA as template, we subsequently cloned the cDNA of this receptor. The receptor cDNA codes for a protein that is strongly related to the first Drosophila receptor (60% amino acid sequence identity in the transmembrane region; 47% identity in the overall sequence) and that is, therefore, most likely to be a second Drosophila allatostatin receptor (named DAR-2). The DAR-2 gene has three introns and four exons. Two of these introns coincide with two introns in the first Drosophila receptor (DAR-1) gene, and have the same intron phasing, showing that the two receptor genes are clearly evolutionarily related. The DAR-2 gene is located at the right arm of the third chromosome, position 98 D-E. This is the first report on the existence of two different allatostatin receptors in an animal.

The Wnt family of secreted signalling molecules are essential in embryo development and tumour formation. The Frizzled (Fz) family of serpentine receptors function as Wnt receptors, but how Fz proteins transduce signalling is not understood. In Drosophila, arrow phenocopies the wingless (DWnt-1) phenotype, and encodes a transmembrane protein that is homologous to two members of the mammalian low-density lipoprotein receptor (LDLR)-related protein (LRP) family, LRP5 and LRP6 (refs 12-15). Here we report that LRP6 functions as a co-receptor for Wnt signal transduction. In Xenopus embryos, LRP6 activated Wnt-Fz signalling, and induced Wnt responsive genes, dorsal axis duplication and neural crest formation. An LRP6 mutant lacking the carboxyl intracellular domain blocked signalling by Wnt or Wnt-Fz, but not by Dishevelled or beta-catenin, and inhibited neural crest development. The extracellular domain of LRP6 bound Wnt-1 and associated with Fz in a Wnt-dependent manner. Our results indicate that LRP6 may be a component of the Wnt receptor complex.

Little is known about the molecular mechanisms of taste perception in animals, particularly the initial events of taste signaling. A large and diverse family of seven transmembrane domain proteins was identified from the Drosophila genome database with a computer algorithm that identifies proteins on the basis of structure. Eighteen of 19 genes examined were expressed in the Drosophila labellum, a gustatory organ of the proboscis. Expression was not detected in a variety of other tissues. The genes were not expressed in the labellum of a Drosophila mutant, pox-neuro70, in which taste neurons are eliminated. Tissue specificity of expression of these genes, along with their structural similarity, supports the possibility that the family encodes a large and divergent family of taste receptors.

Xenopus frizzled 7 can act in canonical and non-canonical Wnt signaling pathways: implications on early patterning and morphogenesis.

Mech Dev. 2000; 92: 227-37

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Here we report the cloning of a Xenopus frizzled transmembrane receptor, Xfz7, and describe its expression pattern during early embryogenesis. Xfz7 mRNA is provided maternally and zygotic transcription peaks in gastrula stages. At that time, transcripts are preferentially localized to the marginal zone and become restricted to distinct regions of the tadpoles in tailbud stages. Overexpression of Xfz7 in embryos perturbs the morphogenesis of trunk and tail, blocks convergence-extension movements in animal caps induced with activin and dorsal lip explants and decreases cadherin-mediated cell adhesion. Xfz7 can interact specifically with Xwnt-8b and signal in the canonical, dorsalizing Wnt pathway. Overexpression of Xfz7 does not trigger the Wnt-1-type pathway but acts in a non-canonical Wnt or morphogenetic-effector pathway involving the activation of protein kinase C (PKC). Xfz7 seems to be involved in different aspects of Wnt signaling during the course of embryogenesis.

Frizzled receptors are components of the Wnt signalling pathway, but how they activate the canonical Wnt/beta-catenin pathway is not clear. Here we use three distinct vertebrate frizzled receptors (Xfz3, Xfz4 and Xfz7) and describe whether and how their C-terminal cytoplasmic regions transduce the Wnt/beta-catenin signal. We show that Xfz3 activates this pathway in the absence of exogenous ligands, while Xfz4 and Xfz7 interact with Xwnt5A to activate this pathway. Analysis using chimeric receptors reveals that their C-terminal cytoplasmic regions are functionally equivalent in Wnt/beta-catenin signalling. Furthermore, a conserved motif (Lys-Thr-X-X-X-Trp) located two amino acids after the seventh transmembrane domain is required for activation of the Wnt/beta-catenin pathway and for membrane relocalization and phosphorylation of Dishevelled. Frizzled receptors with point mutations affecting either of the three conserved residues are defective in Wnt/beta-catenin signalling. These findings provide functional evidence supporting a role of this conserved motif in the modulation of Wnt signalling. They are consistent with the genetic features exhibited by Drosophila Dfz3 and Caenorhabditis elegans mom-5 in which the tryptophan is substituted by a tyrosine.

The beta-catenin signal transduction pathway, which can be activated by secreted Wnt proteins, plays a key role in normal embryonic development and in malignant transformation of the mammary gland and colon. Here we demonstrate, for the first time, that Wnt and beta-catenin signaling also function in cells of the vasculature. RT-PCR analysis showed that primary endothelial and smooth muscle cell cultures, of both mouse and human origin, express members of the Wnt and Wnt receptor (Frizzled) gene families. Transfection of an expression vector for Wnt-1 into primary endothelial cells increased both the free pool of beta-catenin and the transcription from a Lef/tcf-dependent reporter gene construct. Expression of Wnt-1, but not Wnt-5a, also stimulated proliferation of primary endothelial cell cultures. These data show that Wnt and Frizzled proteins can regulate signal transduction, via beta-catenin, in endothelial cells. These findings suggest that Wnt signaling may feature in normal differentiation of the vasculature as well as in pathological settings where endothelial and smooth muscle proliferation is disturbed.

The C-terminal domain of armadillo binds to hypophosphorylated teashirt to modulate wingless signalling in Drosophila.

EMBO J. 1999; 18: 2208-17

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Wnt signalling is a key pathway for tissue patterning during animal development. In Drosophila, the Wnt protein Wingless acts to stabilize Armadillo inside cells where it binds to at least two DNA-binding factors which regulate specific target genes. One Armadillo-binding protein in Drosophila is the zinc finger protein Teashirt. Here we show that Wingless signalling promotes the phosphorylation and the nuclear accumulation of Teashirt. This process requires the binding of Teashirt to the C-terminal end of Armadillo. Finally, we present evidence that the serine/threonine kinase Shaggy is associated with Teashirt in a complex. We discuss these results with respect to current models of Armadillo/beta-catenin action for the transmission of the Wingless/Wnt pathway.

The Delta/Serrate/LAG-2 (DSL) domain containing proteins are considered to be ligands for Notch receptors. However, the physical interaction between DSL proteins and Notch receptors is poorly understood. In this study, we cloned a cDNA for mouse Jagged1 (mJagged1). To identify the receptor interacting with mJagged1 and to gain insight into its binding characteristics, we established two experimental systems using fusion proteins comprising various extracellular parts of mJagged1, a "cell" binding assay and a "solid-phase" binding assay. mJagged1 physically bound to mouse Notch2 (mNotch2) on the cell surface and to a purified extracellular portion of mNotch2, respectively, in a Ca(2+)-dependent manner. Scatchard analysis of mJagged1 binding to BaF3 cells and to the soluble Notch2 protein demonstrated dissociation constants of 0.4 and 0.7 nM, respectively, and that the number of mJagged1-binding sites on BaF3 is 5,548 per cell. Furthermore, deletion mutant analyses showed that the DSL domain of mJagged1 is a minimal binding unit and is indispensable for binding to mNotch2. The epidermal growth factor-like repeats of mJagged1 modulate the affinity of the interaction, with the first and second repeats playing a major role. Finally, solid-phase binding assay showed that Jagged1 binds to Notch1 and Notch3 in addition to Notch2, suggesting that mJagged1 is a ligand for multiple Notch receptors.

The Wnt family of secreted polypeptides participate in a variety of developmental processes in which embryonic polarity is established. To study a role for Wnt ligands in vertebrate axis determination, we interfered with Wnt signaling in the embryo using the extracellular domain of Xenopus Frizzled 8 (ECD8), which blocks Wnt-dependent activation of a target gene in Xenopus ectodermal explants. Expression of ECD8 in ventral blastomeres resulted in formation of secondary axes containing abundant notochord and head structures. These results suggest that Wnt signaling is required to maintain ventral cell fates and has to be suppressed for dorsal development to occur.

Molecular cloning of Frizzled-10, a novel member of the Frizzled gene family.

Biochem Biophys Res Commun. 1999; 262: 39-43

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The Frizzled genes encode WNT receptors. Frizzled-10 (FZD10), a novel member of the Frizzled gene family, has been cloned and characterized. Nucleotide sequence analysis showed that human FZD10 gene encodes a seven-transmembrane-receptor of 581 amino acids, with the N-terminal cysteine-rich domain and the C-terminal Ser/Thr-Xxx-Val motif. Larger amounts of FZD10 mRNA, 4.0 kb in size, were detected in the placenta and fetal kidney, followed by fetal lung and brain. In adult brain, FZD10 mRNA was abundant in the cerebellum. Among cancer cell lines, FZD10 was highly expressed in a cervical cancer cell line, HeLa S3, and moderately in a colon cancer cell line, SW480. The FZD10 gene was mapped to human chromosome 12q24.33. FZD10 shares 65.7% amino-acid identity with Frizzled-9 (FZD9). FZD10 and FZD9 constitute a subfamily among the Frizzled genes.

Characterization and expression pattern of the frizzled gene Fzd9, the mouse homolog of FZD9 which is deleted in Williams-Beuren syndrome.

Genomics. 1999; 57: 235-48

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The frizzled gene family is conserved from insects to mammals and codes for putative Wnt receptors that share a cysteine-rich extracellular domain and seven transmembrane domains. We previously identified a novel frizzled gene, FZD3, now renamed FZD9, in the Williams-Beuren syndrome (WBS) deletion region at chromosomal band 7q11.23 and showed that its product can interact with the Drosophila wingless protein. Here, we report the characterization of the mouse homolog Fzd9. The Fzd9 gene produces a 2.4-kb transcript encoding a 592-amino-acid protein with 95% identity to the human FZD9. Fzd9 was mapped to the conserved syntenic region on distal mouse chromosome 5. By RNA in situ hybridization studies of whole-mount embryos and sections we delineated the temporal and spatial expression patterns in the neural tube, trunk skeletal muscle precursors (myotomes), limb skeletal anlagen, craniofacial regions, and nephric ducts. In adult mouse tissue, the Fzd9 transcript is abundantly present in heart, brain, testis, and skeletal muscle. In testis, Fzd9 is expressed in all spermatogenic cell types. Immunohistochemical studies of cells transfected with a Fzd9 expression construct confirm that Fzd9 is a membrane protein. These results suggest potential Wnt ligands of Fzd9, a role of Fzd9 in skeletal muscle specification, and contributions of FZD9 to the WBS phenotype.

The Drosophila tissue polarity gene starry night encodes a member of the protocadherin family.

Development. 1999; 126: 5421-9

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The tissue polarity genes control the polarity of hairs, bristles and ommatidia in the adult epidermis of Drosophila. We report here the identification of a new tissue polarity gene named starry night (stan). Mutations in this essential gene alter the polarity of cuticular structures in all regions of the adult body. The detailed polarity phenotype of stan on the wing suggested that it is most likely a component of the frizzled (fz) pathway. Consistent with this hypothesis, stan appears to be downstream of and required for fz function. We molecularly cloned stan and found that it encodes a huge protocadherin containing nine cadherin motifs, four EGF-like motifs, two laminin G motifs, and seven transmembrane domains. This suggests that Stan functions in signal reception, perhaps together with Fz.

Wingless transduction by the Frizzled and Frizzled2 proteins of Drosophila.

Development. 1999; 126: 5441-52

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Wingless (Wg) protein is a founding member of the Wnt family of secreted proteins which have profound organizing roles in animal development. Two members of the Frizzled (Fz) family of seven-pass transmembrane proteins, Drosophila Fz and Fz2, can bind Wg and are candidate Wg receptors. However, null mutations of the fz gene have little effect on Wg signal transduction and the lack of mutations in the fz2 gene has thus far prevented a rigorous examination of its role in vivo. Here we describe the isolation of an amber mutation of fz2 which truncates the coding sequence just after the amino-terminal extracellular domain and behaves genetically as a loss-of-function allele. Using this mutation, we show that Wg signal transduction is abolished in virtually all cells lacking both Fz and Fz2 activity in embryos as well as in the wing imaginal disc. We also show that Fz and Fz2 are functionally redundant: the presence of either protein is sufficient to confer Wg transducing activity on most or all cells throughout development. These results extend prior evidence of a ligand-receptor relationship between Wnt and Frizzled proteins and suggest that Fz and Fz2 are the primary receptors for Wg in Drosophila.

Wingless signaling in the Drosophila embryo: zygotic requirements and the role of the frizzled genes.

Development. 1999; 126: 577-86

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Wingless signaling plays a central role during epidermal patterning in Drosophila. We have analyzed zygotic requirements for Wingless signaling in the embryonic ectoderm by generating synthetic deficiencies that uncover more than 99% of the genome. We found no genes required for initial wingless expression, other than previously identified segmentation genes. In contrast, maintenance of wingless expression shows a high degree of zygotic transcriptional requirements. Besides known genes, we have identified at least two additional genomic regions containing new genes involved in Wingless maintenance. We also assayed for the zygotic requirements for Wingless response and found that no single genomic region was required for the cytoplasmic accumulation of Armadillo in the receiving cells. Surprisingly, embryos homozygously deleted for the candidate Wingless receptor, Dfrizzled2, showed a normal Wingless response. However, the Armadillo response to Wingless was strongly reduced in double mutants of both known members of the frizzled family in Drosophila, frizzled and Dfrizzled2. Based on their expression pattern during embryogenesis, different Frizzled receptors may play unique but overlapping roles in development. In particular, we suggest that Frizzled and Dfrizzled2 are both required for Wingless autoregulation, but might be dispensable for late Engrailed maintenance. While Wingless signaling in embryos mutant for frizzled and Dfrizzled2 is affected, Wingless protein is still internalized into cells adjacent to wingless-expressing cells. Incorporation of Wingless protein may therefore involve cell surface molecules in addition to the genetically defined signaling receptors of the frizzled family.

sFRP-2 is a target of the Wnt-4 signaling pathway in the developing metanephric kidney.

Dev Dyn. 1998; 213: 440-51

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Members of the Wnt family of secreted glycoproteins act as short-range signaling molecules in vertebrate embryogenesis. Previous work has shown that Wnt-4 is required for kidney development. Mice lacking functional Wnt-4 fail to form pretubular cell aggregates. Wnt-4 acts as an autoinducer of the mesenchymal to epithelial transition underlying nephron development. We have identified a member of the gene family encoding secreted frizzled related proteins (sFRP), putative Wnt antagonists, that shows overlapping expression with Wnt-4 in aggregating mesenchyme and simple epithelial bodies during metanephric development. sFRP-2 expression is absent in metanephric mesenchyme of kidneys mutant for Wnt-4 and is coinduced with Wnt-4 in isolated metanephric mesenchyme by cells expressing Wnt-4. The cysteine-rich domain of sFRP-2 binds to Wnt-4 as shown by coimmunoprecipitation experiments. Hence, sFRP-2 is a target of the Wnt-4 signaling pathway in the metanephric kidney and may modulate Wnt-4 signaling. sFRP-2 expression is highly dynamic and specific during other aspects of embryogenesis. sFRP-2 is expressed in subpopulations of ependymal cells in spinal cord and brain, in the developing eye, in limb bud mesenchyme, in the heart, and strongly in skeletogenic condensations of facial bones, suggesting widespread interaction with other members of the Wnt gene family during embryogenesis.

frizzled and frizzled 2 play a partially redundant role in wingless signaling and have similar requirements to wingless in neurogenesis.

Cell. 1998; 95: 1027-36

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The Drosophila Frizzled (Fz) and Frizzled2 (DFz2) proteins function as receptors for Wingless (Wg) in tissue culture cells. While previous results indicate that loss of function for fz has tissue polarity defects, loss-of-function effects of Dfz2 are not known. Here, we have examined the requirements of fz and Dfz2 during neurogenesis. Our results indicate that both Fz and DFz2 function in Wg signaling, and loss of either of the two affects the same subset of neuroblasts as those affected by loss of wg. While these defects are partially penetrant in embryos lacking either fz or Dfz2, the penetrance is significantly enhanced in embryos lacking both. Since the penetrance of the CNS phenotypes is not complete in double mutants, additional components that allow some degree of Wg signaling must exist in vivo.

Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction.

Nature. 1998; 391: 357-62

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The Spemann organizer in amphibian embryos is a tissue with potent head-inducing activity, the molecular nature of which is unresolved. Here we describe dickkopf-1 (dkk-1), which encodes Dkk-1, a secreted inducer of Spemann's organizer in Xenopus and a member of a new protein family. Injections of mRNA and antibody indicate that dkk-1 is sufficient and necessary to cause head induction. dkk-1 s a potent antagonist of Wnt signalling, suggesting that dkk genes encode a family of secreted Wnt inhibitors.

An expression cloning screen was used to isolate a novel gene homologous to the extracellular cysteine-rich domain of frizzled receptors. The gene (which we called sizzled for secreted frizzled) was shown to encode a soluble secreted protein, containing a functional signal sequence but no transmembrane domains. Sizzled (szl) is capable of inhibiting Xwnt8 as assayed by (1) dose-dependent inhibition of siamois induction by Xwnt8 in animal caps, (2) rescue of embryos ventralized by Xwnt8 DNA and (3) inhibition of XmyoD expression in the marginal zone. Szl can dorsalize Xenopus embryos if expressed after the midblastula transition, strengthening the idea that zygotic expression of wnts and in particular of Xwnt8 plays a role in antagonizing dorsal signals. It also suggests that inhibiting ventralizing wnts parallels the opposition of BMPs by noggin and chordin. szl expression is restricted to a narrow domain in the ventral marginal zone of gastrulating embryos. szl thus encodes a secreted antagonist of wnt signaling likely involved in inhibiting Xwnt8 and XmyoD ventrally and whose restricted expression represents a new element in the molecular pattern of the ventral marginal zone.

From nematode to man, the transmembrane receptors of the Notch family act throughout embryonic and post-embryonic development to regulate the acquisition and/or maintenance of specific differentiative states. We will review here our current state of knowledge on Notch receptors structure and signalling activity.

Identification and characterization of a seven transmembrane hormone receptor using differential display.

Genomics. 1997; 41: 301-8

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Targeted mutagenesis analysis has shown that the Cmyb proto-oncogene, which encodes a sequence-specific DNA binding protein, is required for normal murine fetal liver erythropoiesis and myelopoiesis. To identify novel genes involved in hematopoiesis, differential display analysis was conducted using total liver RNA isolated from 14.5-day postcoitus Cmyb wildtype, heterozygous, and homozygous mutant littermates. Using 4 oligo(dT) 3' primers and 5 arbitrary decamers as 5' primers, 22 differentially expressed genes have been identified. Eight putatively novel genes were identified from 12 cDNAs that were sequenced. One gene, initially designated DD7A5-7, is primarily expressed in cells of the myeloid lineage. The full-length DD7A5-7 cDNA is 3239 nucleotides, encoding a putative protein of 931 amino acids. The protein is a member of a family of hormone receptors containing 7 transmembrane segments. The receptor also contains 7 epidermal growth factor-like (Egf-like) motifs at the amino terminal of the predicted protein. The gene is alternatively spliced, resulting in the deletion of one or more copies of the Egf-like motif. DD7A5-7 maps to mouse Chromosome 17 and is the putative homologue of EMR1, a recently described Egf-like module containing mucin-like hormone receptor with 7 transmembrane segments in humans. Our results indicate that the Cmyb mutant fetuses represent a unique resource for identifying genes involved in hematopoiesis.

Head induction by simultaneous repression of Bmp and Wnt signalling in Xenopus.

Nature. 1997; 389: 517-9

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The Spemann organizer of the amphibian embryo can be subdivided into two discrete activities, namely trunk organizer and head organizer. Several factors secreted from the organizer that are involved in trunk organization are thought to act by repressing Bmp signalling. With the exception of the secreted factor cerberus, little is known about head-organizer inducers. Here we show that co-expression of a dominant-negative Bmp receptor with inhibitors of the Wnt-signalling pathway in Xenopus leads to the induction of complete secondary axes, including a head. This induction does not require expression of the siamois marker of Nieuwkoop centre signalling, suggesting that cells are directly shifting to head-organizer fate. Furthermore, we find that cerberus is a potent inhibitor of Wnt signalling. Our results indicate that head-organizer activity results from the simultaneous repression of Bmp and Wnt signalling and they suggest a mechanism for region-specific induction by the organizer.

Signaling molecules of the Wnt gene family are involved in the regulation of dorso-ventral, segmental and tissue polarity in Xenopus and Drosophila embryos. Members of the frizzled gene family, such as Drosophila frizzled-2 and rat frizzled-1, have been shown encode Wnt binding activity and to engage intracellular signal transduction molecules known to be part of the Wnt signaling pathway. Here we describe the cloning and characterization of Fritz, a mouse (mfiz) and human (hfiz) gene which codes for a secreted protein that is structurally related to the extracellular portion of the frizzled genes from Drosophila and vertebrates. The Fritz protein antagonizes Wnt function when both proteins are ectopically expressed in Xenopus embryos. In early gastrulation, mouse fiz mRNA is expressed in all three germ layers. Later in embryogenesis fiz mRNA is found in the central and peripheral nervous systems, nephrogenic mesenchyme and several other tissues, all of which are sites where Wnt proteins have been implicated in tissue patterning. We propose a model in which Fritz can interfere with the activity of Wnt proteins via their cognate frizzled receptors and thereby modulate the biological responses to Wnt activity in a multitude of tissue sites.

A novel human homologue of the Drosophila frizzled wnt receptor gene binds wingless protein and is in the Williams syndrome deletion at 7q11.23.

Hum Mol Genet. 1997; 6: 465-72

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Williams syndrome (WS) is a developmental disorder with a characteristic personality and cognitive profile that is associated, in most cases, with a 2 Mb deletion of part of chromosome band 7q11.23. By applying CpG island cloning methods to cosmids from the deletion region, we have identified a new gene, called FZD3. Dosage blotting of DNA from 11 WS probands confirmed that it is located within the commonly deleted region. Sequence comparisons revealed that FZD3, encoding a 591 amino acid protein, is a novel member of a seven transmembrane domain receptor family that are mammalian homologs of the Drosophila tissue polarity gene frizzled. FZD3 is expressed predominantly in brain, testis, eye, skeletal muscle and kidney. Recently, frizzled has been identified as the receptor for the wingless (wg) protein in Drosophila. We show that Drosophila as well as human cells, when transfected with FZD3 expression constructs, bind Wg protein. In mouse, the wg homologous Wnt1 gene is involved in early development of a large domain of the central nervous system encompassing much of the midbrain and rostral metencephalon. The potential function of FZD3 in transmitting a Wnt protein signal in the human brain and other tissues suggests that heterozygous deletion of the FZD3 gene could contribute to the WS phenotype.

Frzb-1 is a secreted antagonist of Wnt signaling expressed in the Spemann organizer.

Cell. 1997; 88: 747-56

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Frzb-1 is a secreted protein containing a domain similar to the putative Wnt-binding region of the frizzled family of transmembrane receptors. Frzb-1 is widely expressed in adult mammalian tissues. In the Xenopus gastrula, it is expressed and regulated as a typical Spemann organizer component. Injection of frzb-1 mRNA blocks expression of XMyoD mRNA and leads to embryos with enlarged heads and shortened trunks. Frzb-1 antagonizes the effects of Xwnt-8 ectopic expression in a non-cell-autonomous manner. Cultured cells transfected with a membrane-tethered form of Wnt-1 bind epitope-tagged Frzb-1 in the 10(-10) M range. The results strengthen the view that the Spemann organizer is a source of secreted inhibitory factors.

Purification and molecular cloning of a secreted, Frizzled-related antagonist of Wnt action.

Proc Natl Acad Sci U S A. 1997; 94: 6770-5

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Frizzled polypeptides are integral membrane proteins that recently were shown to function as receptors for Wnt signaling molecules. Here, we report the identification of a novel, secreted 36-kDa protein that contains a region homologous to a putative Wnt-binding domain of Frizzleds. This protein, called Frizzled-related protein (FRP), was first identified as a heparin-binding polypeptide that copurified with hepatocyte growth factor/scatter factor in conditioned medium from a human embryonic lung fibroblast line. Degenerate oligonucleotides, based on the NH2-terminal sequence of the purified protein, were used to isolate corresponding cDNA clones. These encoded a 313-amino acid polypeptide, containing a cysteine-rich domain of approximately 110 residues that was 30-40% identical to the putative ligand-binding domain of Frizzled proteins. A 4.4-kb transcript of the FRP gene is present in many organs, both in the adult and during embryogenesis, and homologs of the gene are detectable in DNA from several vertebrate species. In biosynthetic studies, FRP was secreted but, like Wnts, tended to remain associated with cells. When coexpressed with several Wnt family members in early Xenopus embryos, FRP antagonized Wnt-dependent duplication of the embryonic dorsal axis. These results indicate that FRP may function as an inhibitor of Wnt action during development and in the adult.

The frizzled (fz) gene of Drosophila is essential for the development of normal tissue polarity in the adult cuticle of Drosophila. In fz mutants the parallel array of hairs and bristles that decorate the cuticle is disrupted. Previous studies have shown the fz encodes a membrane protein with seven putative transmembrane domains, and that it has a complex role in the development of tissue polarity, as there exist both cell-autonomous and cell nonautonomous alleles. We have now examined a larger number of alleles and found that 15 of 19 alleles display cell nonautonomy. We have examined these and other alleles by Western blot analysis and found that most fz mutations result in altered amounts of Fz protein, and many also result in a Fz protein that migrates aberrantly in SDS-PAGE. We have sequenced a subset of these alleles. Cell nonautonomous fz alleles were found to be associated with mutations that altered amino acids in all regions of the Fz protein. Notably, the four cell-autonomous mutations were all in a proline residue located in the presumptive first cytoplasmic loop of the protein. We have also cloned and sequenced the fz gene from D. virilis. Conceptual translation of the D. virilis open reading frame indicates that the Fz protein is unusually well conserved. Indeed, in the putative cytoplasmic domains the Fz proteins of the two species are identical.

The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the hedgehog signal.

Cell. 1996; 86: 221-32

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Smoothened (smo) is a segment polarity gene required for correct patterning of every segment in Drosophila. The earliest defect in smo mutant embryos is loss of expression of the Hedgehog-responsive gene wingless between 1 and 2 hr after gastrulation. Since smo mutant embryos cannot respond to exogenous Hedgehog (Hh) but can respond to exogenous Wingless, the smo product functions in Hh signaling. Smo acts downstream of or in parallel to Patched, an antagonist of the Hh signal. The smo gene encodes an integral membrane protein with characteristics of G protein-coupled receptors and shows homology to the Drosophila Frizzled protein. Based on its predicted physical characteristics and on its position in the Hh signaling pathway, we suggest that smo encodes a receptor for the Hh signal.

Secreted proteins of the Hedgehog (Hh) family have diverse organizing roles in animal development. Recently, a serpentine protein Smoothened (Smo) has been proposed as a Hh receptor. Here, we present evidence that implicates another multiple-pass transmembrane protein, Patched (Ptc), in Hh reception and suggests a novel signal transduction mechanism in which Hh binds to Ptc, or a Ptc-Smo complex, and thereby induces Smo activity. Our results also show that Ptc limits the range of Hh action; we provide evidence that high levels of Ptc induced by Hh serve to sequester any free Hh and therefore create a barrier to its further movement.

BACKGROUND: Wnts are secreted proteins implicated in cell-cell interactions during embryogenesis and tumorigenesis, but receptors involved in transducing Wnt signals have not yet been definitively identified. Members of a large family of putative transmembrane receptors homologous to the frizzled protein in Drosophila have been identified recently in both vertebrates and invertebrates, raising the question of whether they are involved in transducing signals for any known signaling factors. RESULTS: To test the potential involvement of frizzled homologs in Wnt signaling, we examined the effects of overexpressing rat frizzled-1 (Rfz-1) on the subcellular distribution of Wnts and of dishevelled, a cytoplasmic component of the Wnt signalling pathway. We demonstrate that ectopic expression of Rfz-1 recruits the dishevelled proten-as well as Xenopus Wnt-8 (Xwnt-8), but not the functionally distinct Xwnt-5A-to the plasma membrane. Moreover, Rfz-1 is sufficient to induce the expression of two Xwnt-8-responsive genes, siamois and Xnr-3, in Xenopus explants in a manner which is antagonized by glycogen synthase kinase-3, which also antagonizes Wnt signaling. When Rfz-1 and Xwnt-8 are expressed together in this assay, we observe greater induction of these genes, indicating that Rfz-1 can synergize with a Wnt. CONCLUSIONS: The results demonstrate that a vertebrate frizzled homolog is involved in Wnt signaling in a manner which discriminates between functionally distinct Wnts, which involves translocation of the dishevelled protein to the plasma membrane, and which works in a synergistic manner with Wnts to induce gene expression. These data support the likely function of frizzled homologs as Wnt receptors, or as components of a receptor complex.

Members of the highly conserved Wnt gene family serve key functions in cellular growth and differentiation. Regulated expression of the murine Wnt-1 proto-oncogene is essential for normal development of the embryonic nervous system and when misexpressed in the mammary gland leads to hyperplasias and tumorigenic progression. Wnt-1 encodes a cysteine rich glycoprotein that is secreted and tightly associated with the cell surface and/or extracellular matrix. This restricted diffusion of Wnt-1 protein is important for specifying a spatially defined pattern of Wnt-activity necessary for cell to cell signaling in cell growth and differentiation. Since the receptors and cell surface molecules that bind Wnt proteins are as yet unknown we have sought to define the sequences within Wnt-1 protein that enable cell surface association and to define the nature of the cell surface structures with which Wnt-1 protein associates. First, site directed mutagenesis was used to identify a basic amino acid sequence motif within Wnt-1 protein that is required for this protein to accumulate on the cell surface. Second, wild type Wnt-1 protein was expressed in a series of proteoglycan-deficient CHO cell lines to show that, contrary to expectations, cellular glycosaminoglycans are not required for processing, secretion, cell surface association and accumulation of Wnt-1 protein.

The protein Sonic hedgehog (Shh) controls patterning and growth during vertebrate development. Here we demonstrate that it binds Patched (vPtc), which has been identified as a tumour-suppressor protein in basal cell carcinoma, with high affinity. We show that Ptc can form a physical complex with a newly cloned vertebrate homologue of the Drosophila protein Smoothened (vSmo), and that vSmo is coexpressed with vPtc in many tissues but does not bind Shh directly. These findings, combined with available genetic evidence from Drosophila, support the hypothesis that Ptc is a receptor for Shh, and that vSmo could be a signalling component that is linked to Ptc.

Distinct cellular and subcellular patterns of expression imply distinct functions for the Drosophila homologues of moesin and the neurofibromatosis 2 tumor suppressor, merlin.

J Cell Biol. 1996; 133: 843-52

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Interest in members of the protein 4.1 super-family, which includes the ezrin-radixin-moesin (ERM) group, has been stimulated recently by the discovery that the human neurofibromatosis 2 (NF2) tumor suppressor gene encodes an ERM-like protein, merlin. Although many proteins in this family are thought to act by linking the actin-based cytoskeleton to transmembrane proteins, the cellular functions of merlin have not been defined. To investigate the cellular and developmental functions of these proteins, we have identified and characterized Drosophila homologues of moesin (Dmoesin) and of the NF2 tumor suppressor merlin (Dmerlin). Using specific antibodies, we show that although these proteins are frequently coexpressed in developing tissues, they display distinct subcellular localizations. While Dmoesin is observed in continuous association with the plasma membrane, as is typical for an ERM family protein, Dmerlin is found in punctuate structures at the membrane and in the cytoplasm. Investigation of Dmerlin cultured cells demonstrates that it is associated with endocytic compartments. As a result of these studies, we propose that the merlin protein has unique functions in the cell which differ from those of other ERM family members.

Molecular cloning and function expression of a diuretic hormone receptor from the house cricket, Acheta domesticus.

Insect Biochem Mol Biol. 1996; 26: 1-6

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Insect diuretic hormones regulate fluid and ion secretion and the receptors with which they interact are attractive targets for new insect control agents. Recently, a diuretic hormone receptor from the moth Manduca sexta was isolated by expression cloning and found to be a member of the calcitonin/secretin/corticotropin releasing factor family of G-protein coupled receptors [Reagan J. D. (1994) J. Biol. Chem. 269, 9-12]. Degenerate oligonucleotides were designed based upon conserved regions in this receptor family and used to isolate a diuretic hormone receptor from the house cricket, Acheta domesticus. The complementary DNA isolated encodes a protein consisting of 441 amino acids with seven putative membrane spanning regions. Interestingly, unlike the M. sexta diuretic hormone receptor, the cricket diuretic hormone receptor contains a putative signal sequence. The receptor shares 53% and 38% sequence identity with the M. sexta diuretic hormone and human corticotropin releasing factor receptors respectively. When expressed in COS-7 cells, the receptor binds A. domesticus diuretic hormone with high affinity and stimulates adenylate cyclase with high potency. Four other insect diuretic hormones are considerably less effective at stimulating adenylate cyclase in COS-7 cells transfected with the receptor. This is in contrast to the M. sexta diuretic hormone receptor which is stimulated by all five insect diuretic hormones with high potency.

Drosophila hormone receptor 38: a second partner for Drosophila USP suggests an unexpected role for nuclear receptors of the nerve growth factor-induced protein B type.

Proc Natl Acad Sci U S A. 1995; 92: 7966-70

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In Drosophila the response to the hormone ecdysone is mediated in part by Ultraspiracle (USP) and ecdysone receptor (EcR), which are members of the nuclear receptor superfamily. Heterodimers of these proteins bind to ecdysone response elements (EcREs) and ecdysone to modulate transcription. Herein we describe Drosophila hormone receptor 38 (DHR38) and Bombyx hormone receptor 38 (BHR38), two insect homologues of rat nerve growth factor-induced protein B (NGFI-B). Although members of the NGFI-B family are thought to function exclusively as monomers, we show that DHR38 and BHR38 in fact interact strongly with USP and that this interaction is evolutionarily conserved. DHR38 can compete in vitro against EcR for dimerization with USP and consequently disrupt EcR-USP binding to an EcRE. Moreover, transfection experiments in Schneider cells show that DHR38 can affect ecdysone-dependent transcription. This suggests that DHR38 plays a role in the ecdysone response and that more generally NGFI-B type receptors may be able to function as heterodimers with retinoid X receptor type receptors in regulating transcription.

The appearance of puffs on the polytene chromosomes of insect salivary glands incubated with 20-hydroxyecdysone provided the first demonstration that steroids act directly at the gene transcriptional level to bring about subsequent cellular changes (Becker, 1959; Clever and Karlson, 1960). Despite that auspicious beginning, learning about the molecular mechanisms that underlie the hormonal regulation of insect development was impeded for many years by the difficulty associated with isolating and identifying rare regulatory factors from limited tissue sources. The advent of recombinant DNA methodology and powerful techniques such as the polymerase chain reaction (PCR) along with the recognition that many important endocrine factors are structurally conserved across a wide range of species has, however, all but eliminated the technical obstacles once facing the insect endocrinologist trying to isolate and study these regulatory molecules. This review will discuss recent progress and recall some earlier experiments concerning the molecular basis of hormonal action in insects focusing primarily on the members of the nuclear hormone receptor superfamily in Drosophila melanogaster. Two members of this family comprise the functional ecdysteroid receptor and at least a dozen other "orphans" have been identified in Drosophila for which no cognate ligand has yet been found. Many of these orphans are regulated by ecdysteroids. A discussion of juvenile hormone binding proteins that are not family members has been included because of their potential impact on nuclear receptor function. As receptor homologues have been identified in other insects, several general ideas concerning insect hormonal regulation have begun to emerge and these will be examined from a comparative point of view.

Identification, characterization, and developmental regulation of a receptor guanylyl cyclase expressed during early stages of Drosophila development.

J Biol Chem. 1995; 270: 7189-96

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Membrane forms of guanylyl cyclase are single-transmembrane proteins that are activated by the binding of specific peptide ligands to their extracellular domains. In this report, we describe the identification and characterization of a Drosophila cDNA clone encoding a protein, DrGC-1, with high sequence identity to members of this family of receptor proteins. The protein contains a single, hydrophobic domain predicted to represent a transmembrane segment separating an extracellular domain with significant sequence identity (30%) to sea urchin egg peptide receptors from intracellular domains containing a protein kinase-like domain followed by a region with high sequence identity (65%) to cyclase catalytic domains found in receptor guanylyl cyclases from both vertebrates and invertebrates. In contrast to other members of this family, DrGC-1 is predicted to contain a carboxyl-terminal extension of 430 residues that has no homology to any described protein. Northern analysis indicates that DrGC-1 transcripts are present at variable levels in all stages of development. In situ hybridization demonstrates that high levels of uniformly distributed transcript are present in 0-2-h embryos. Later in embryogenesis (14-18 h), elevated levels of hybridization appear to be preferentially associated with muscle fibers.

Sequence comparisons of vitellogenins from a wide range of organisms have identified regions of similarity not only to each other but also to vertebrate apolipoproteins (e.g. apoB-100 and apoE). Furthermore, the chicken vitellogenin receptor, which also binds apolipoproteins receptor (LDLR) superfamily [Bujo, H., Hermann, M., Kaderli, M. O., Jacobsen, L., Sugawara, S., Nimpf, J., Yamamoto, T. & Schneider, W. J. (1994) EMBO J. 13, 5165-5175]. The yolk proteins of higher dipterans are exceptional, however, and instead show similarity to lipoprotein lipases. The molecular characterization of the putative Drosophila melanogaster vitellogenin receptor gene, yolkless (yl), described in this report reveals that the protein it encodes (Yl), is also a member of the LDLR superfamily. The ovary-specific 6.5-kb yl RNA codes for a protein of approximately 210 kDa which contains all three motifs common to the LDLR class of proteins. Within this superfamily, Yl may be related more to the LDLR-related proteins (LRPs), which bind both apolipoproteins and lipoprotein lipases. The similarity of Yl to the other LDLR proteins is restricted to the putative extracellular domain. Most noticeably, the cytoplasmic domain of Yl lacks the typical NPXY sequence which is involved in receptor internalization.

The murine Wnt family of proteins consists of at least 12 members that possess significant amino acid homology. Current evidence suggests that these proteins are secreted cell-signaling molecules which are likely to have multiple roles during both embryonic development and oncogenesis. Although the biochemical properties of Wnt-1 have been thoroughly examined, less is known about the characteristics of other Wnt family members. We have compared the properties of six murine Wnt proteins (Wnt-1, Wnt-3a, Wnt-5a, Wnt-5b, Wnt-6, and Wnt-7b) transiently expressed in COS cells. All members enter the endoplasmic reticulum (ER) and are glycosylated. However, all six Wnt proteins are primarily retained in the ER in association with BiP, a resident ER protein that binds to improperly folded proteins and prevents their secretion and/or promotes proper folding. Although all Wnt family members examined are similarly processed, one notable difference was identified. Whereas addition of suramin to COS cell cultures significantly increases the levels of all six Wnts in the medium, the addition of heparin only influences the levels of Wnt-1, Wnt-6, and Wnt-7b.

Interspecific comparisons reveal conserved features of the Drosophila Toll protein.

Gene. 1994; 139: 223-8

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The Toll gene of Drosophila melanogaster produces a transmembrane cell adhesion protein that is required to establish the dorso-ventral axis of the embryo. The Toll protein's extracellular domain contains Leu-rich repeats (LRR), implicated in intermolecular interactions, and its large intracellular domain transduces a signal that eventually reaches the nucleus. Here, we report amino-acid (aa) sequences encoded by the Toll genes of D. pseudoobscura and D. virilis, and two distinct Toll-like genes of the grasshopper, Schistocerca americana. Interspecific comparisons show a Toll-specific subfamily of LRR, and a strikingly high degree of conservation in the cytoplasmic domain. Interestingly, many aa residues conserved among the insect Toll-like cytoplasmic domains are also conserved in mammalian and avian type-I interleukin-1 receptors and the hypothetical product of a transcript, MyD88, found in murine myeloid cells. Thus, we identify a set of conserved aa in the cytoplasmic domain which might be used in a signal-transduction pathway shared by invertebrates and vertebrates.

The Drosophila frizzled (fz) gene is required for the development of normal tissue polarity in the epidermis. Genetic epistasis experiments argue that fz is at the top of a regulatory hierarchy that controls the subcellular site for prehair initiation within the cells of the pupal wing (Wong and Adler, 1993; J. Cell Biol. 123, 209-221). Genetic mosaic experiments indicate that fz has both cell autonomous and cell non-autonomous functions that are separately mutable (Vinson and Adler, 1987; Nature 329, 549-551). Two species of fz mRNA have been identified, raising the question as to whether the two functions are provided by a single protein or by two separate protein species. We generated transgenic flies that express each of these mRNAs under the control of an hsp70 promoter. Only one of the transgenes (hsfzI) showed any fz activity. At 29 degrees C, the hsfzI transgene provided almost complete rescue of a null fz mutation, indicating that the protein encoded by this cDNA can fulfill both fz functions. Overexpression of the hsfzI transgene resulted in two distinct tissue polarity phenotypes depending on the time of heat shock.

Dror, a potential neurotrophic receptor gene, encodes a Drosophila homolog of the vertebrate Ror family of Trk-related receptor tyrosine kinases.

Proc Natl Acad Sci U S A. 1993; 90: 7109-13

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We have identified a Drosophila gene, Dror, which encodes a putative receptor tyrosine kinase (RTK) and maps to cytological location 31B/C on the second chromosome. In embryos, this gene is expressed specifically in the developing nervous system. The Dror protein appears to be a homolog of two human RTKs, Ror1 and Ror2. Dror and Ror1 proteins share 36% amino acid identity in their extracellular domains and 61% identity in their catalytic tyrosine kinase (TK) domains. Ror1 and Ror2 were originally identified on the basis of the similarity of their TK domains to the TK domains of members of the Trk family of neurotrophin receptors. The Dror protein shows even greater similarity to the Trk proteins within this region than do the human Ror proteins. In light of its similarity to trk and its neural-specific expression pattern, we suggest that Dror may encode a neurotrophic receptor that functions during early stages of neural development in Drosophila.

Mammalian integral membrane receptors are homologous to facilitators and antiporters of yeast, fungi, and eubacteria.

Protein Sci. 1993; 2: 20-30

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We demonstrate that three integral membrane receptors of mammals--the ecotropic retroviral leukemia receptor (ERR), the human retroviral receptor (HRR), and the T-cell early activator (Tea)--are homologous to a family of transporters specific for amino acids, polyamines, and choline (APC), which catalyze solute uniport, solute:cation symport, or solute:solute antiport in yeast, fungi, and eubacteria. Interestingly, the ERR membrane protein was recently shown to function as a cation:amino acid cotransporter. A binary sequence similarity matrix and an evolutionary tree of the 14 members of this family, illustrating their sequence similarities and divergences, were constructed. Other proteins, including the developmentally controlled GerAII spore germination protein of Bacillus subtilis and the acetylcholine receptor of Drosophila melanogaster gave sequence comparison scores of a sufficiently large magnitude to suggest (but not to establish) a common evolutionary origin with members of the APC family. We report an extended and corrected Tea cDNA sequence and show that the mammalian Tea and ERR encoding genes are differentially expressed in tissues and cell lines. Furthermore, the two mammalian cDNA sequences hybridize with other vertebrate and yeast genomic DNAs under stringent conditions. These observations support the notion that cell surface receptor proteins in mammals are transport proteins that share a common origin with transport proteins of single-celled organisms. Thus, permeases of essential metabolites may function pathologically as viral receptors.

Isolation and expression of two novel Wnt/wingless gene homologues in Drosophila.

Development. 1992; 115: 475-85

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Wingless (wg), the Drosophila homologue of the mouse Wnt-1 proto-oncogene, is a segment polarity gene essential in each segment for normal Drosophila development. We here report the isolation of two novel Drosophila Wnt homologues, DWnt-2 and DWnt-3, and thus the existence of a Wnt/wingless gene family in Drosophila. DWnt-2 and DWnt-3 map to chromosome 2 position 45E and chromosome X position 17A/B, respectively. DWnt-2 and DWnt-3, like the other known Wnt genes, encode amino-terminal signal peptides suggesting that the gene products are secreted proteins. The putative translation product of DWnt-2 and the carboxy-terminal half of the deduced DWnt-3 product are both rich in conserved cysteine residues. In comparison with other Wnt gene products, mostly about 40 x 10(3) relative molecular mass, the DWnt-3 protein has an extended amino terminus and a long internal insert, and its predicted relative molecular mass is 113 x 10(3). The expression patterns of these two Wnt/wg homologues are dynamic during Drosophila embryogenesis. The distribution of DWnt-2 transcripts is predominantly segmented, with the additional presence of transcripts in the presumptive gonads. Transcripts of both DWnt-2 and DWnt-3 appear to be associated with limb primordia in the embryo and may therefore specify limb development. DWnt-3 is also expressed in mesodermal and neurogenic regions. The distribution of DWnt-3 transcripts in cells of the central nervous system (CNS) during Drosophila embryogenesis suggests that DWnt-3 could be involved in CNS development.

The function of the frizzled (fz) locus in Drosophilia melanogaster is required to coordinate the cytoskeletons of epidermal cells to produce a parallel array of cuticular hairs and bristles (for example on the wild-type wing all hairs point towards the distal tip). In fz mutants it is not the structure of individual hairs and bristles that is altered, but their orientation with respect to their neighbours and the organism as a whole. Mitotic clone analysis indicates that fz has two functions in the developing wing. It is required for the proximal-distal transmission of an intercellular polarity signal, a process that is expected to be at least partly extracellular. It is also required for cells to respond to the polarity signal, which is expected to be a cytoplasmic function. The fz locus could encode either one bifunctional or two single-function proteins. We report here that, in pupae, fz produces a messenger RNA that encodes a protein with seven putative transmembrane domains. Thus, the Fz protein should contain both extracellular and cytoplasmic domains, which could function in the transmission and interpretation of polarity information, respectively. This is the first reported sequence for the protein product of a tissue polarity gene.